Vitamin D status of human immunodeficiency virus–positive patients with advanced liver disease enrolled in the solid organ transplantation in HIV: Multi-site study
Andrea D. Branch,
Division of Liver Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY
Address reprint requests to Andrea D. Branch, Ph.D., Division of Liver Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, Room 11-24, New York, NY 10029. Telephone: 212-659-8371; FAX: 212-849-2574; E-mail: firstname.lastname@example.org
The vitamin D axis plays an integral role in maintaining human health. Vitamin D is synthesized in the skin or acquired from dietary sources and is transported to the liver. There, it is hydroxylated to form 25-hydroxyvitamin D [25(OH)D], which is the single best indicator of a patient's vitamin D status. A second hydroxylation step occurs in the kidneys and at multiple sites of local metabolism to produce 1,25-dihydroxyvitamin D, which acts as a steroid hormone that regulates gene expression in multiple tissues, increases the intestinal absorption of calcium, and strengthens bone. In addition to its classic functions in bone and calcium metabolism, vitamin D has been reported to have many additional effects that may benefit patients undergoing solid organ transplantation, including anti-inflammatory and antifibrotic effects, reduced rates of acute allograft rejection,[3-5] and protection against liver cancer.[6, 7]
Several studies have shown a positive correlation between higher 25(OH)D levels and antiviral treatment responses in hepatitis C virus (HCV)–monoinfected patients[8-12]; however, this relationship may be influenced by racial differences in vitamin D endocrinology. One study found a positive relationship between 25(OH)D levels and sustained virological response (SVR) rates in human immunodeficiency virus (HIV)/HCV–coinfected patients, whereas another study did not find an association with SVR but did find that 25(OH)D levels were negatively correlated with liver fibrosis. Recent studies suggest that the relationship between 25(OH)D levels and treatment response rates may depend on genetic differences in the haplotype of the vitamin D receptor, which may account for some of the differences in the findings of various studies. Vitamin D supplementation has been reported to increase SVR rates in HCV-monoinfected patients undergoing interferon/ribavirin treatment,[17, 18] and it has also been associated with greater SVR rates in HCV-positive patients treated after liver transplantation (LT).
Nutritional deficiencies are common among patients with advanced disease and compromise health across a spectrum of disease conditions. A recent study found that 81% of patients awaiting LT had 25(OH)D levels < 32 ng/mL, which many experts consider to be the lower limit of the optimal level. Adequate levels of vitamin D are particularly important for patients with advanced liver disease because these patients have a high risk of bone fractures. A study of 360 patients who underwent LT found that approximately 20% had evidence of bone fractures before LT, and the fracture rate increased after LT, with 25% of the patients experiencing a new fracture in the 6 months after LT. Vitamin D may be especially important for HIV-positive patients with end-stage liver disease because an HIV infection can directly contribute to bone loss, and the antiretroviral medications used to suppress HIV infections can also cause bone loss and increased bone fractures. HIV practice guidelines recommend a level > 32 ng/mL.
The Solid Organ Transplantation in HIV: Multi-Site Study was performed to assess the safety and outcomes of transplantation in HIV-positive patients since the introduction of highly active antiretroviral therapy. This retrospective, ancillary study was undertaken to determine whether vitamin D needs were met by the standard practices being followed at the 17 academic medical centers participating in the parent study and to assess risk factors for vitamin D deficiency after LT.
PATIENTS AND METHODS
This is a retrospective analysis of data and samples collected during a prospective cohort study of HIV-positive patients with advanced liver disease who participated in the Solid Organ Transplantation in HIV: Multi-Site Study, which was an investigation conducted at 17 centers in the United States (ClinicalTrials.gov identifier NCT00074386). The inclusion criteria for the main study have been described previously. The research protocol was approved and monitored by the institutional review boards of all participating centers and the data and safety monitoring board of the National Institute of Allergy and Infectious Diseases. The study group included in this investigation comprised 154 LT candidates/recipients who had at least 1 serum specimen banked before or after LT that was available for 25(OH)D quantitation via the DiaSorin immunoassay. Specimens were collected between November 2003 and October 2010 and stored in freezers at −80°C. The time points for specimens used in the analysis included the time of enrollment (before transplantation) and 3 months, 6 months, 1 year, and 2 years after transplantation. Forty of the 154 subjects did not undergo LT during the study and hence had baseline samples only. Eighty-three of the 114 LT patients had both pre- and post-LT samples, 15 had post-LT samples only, and 16 had pre-LT samples only. From LT recipients, pre-LT samples were collected at a median of 1 day [interquartile range (IQR) = 0-65 days] before LT. Vitamin D deficiency was defined as a 25(OH)D level < 20 ng/mL, severe deficiency was defined as a level < 10 ng/mL, inadequacy was defined as a level < 32 ng/mL, and potential toxicity was defined as a level > 150 ng/mL. Centers were queried about routine vitamin D supplementation strategies after LT. Center-specific vitamin D supplementation strategies were categorized as either prescribed on an individual basis or routinely prescribed for all patients. After LT, vitamin D supplement use was reported by patients on concomitant medication logs at select time points per the study protocol. Information about pre-LT supplement use was not available.
Treatment of Recurrent HCV
HCV-positive patients who developed cholestatic hepatitis or had evidence of fibrosis progression were treated with pegylated interferon α2a or α2b plus ribavirin (target dose = 800 mg/day) after LT. Recurrence was defined by the presence of cholestatic hepatitis or bridging fibrosis/cirrhosis in a liver biopsy sample, by the initiation of HCV therapy, or by graft loss attributed to HCV.
Continuous variables were analyzed with the Wilcoxon rank-sum test, and paired continuous variables were analyzed with the signed-rank test. Logistic regression was used to identify baseline factors associated with vitamin D deficiency before LT (at enrollment) and vitamin D deficiency and severe vitamin D deficiency 1 year after LT. The analyzed variables included the location of the transplant center (north versus south; Supporting Table 1), season of serum specimen collection (season of measurement), laboratory-based Model for End-Stage Liver Disease (MELD) score, age, race, sex, ethnicity, cirrhosis as the subject's primary hepatic diagnosis (versus cholestatic disease, fulminant liver failure, metabolic disease, tumor, or toxicity), etiology of chronic liver disease (HCV versus no HCV), nonnucleoside reverse transcriptase inhibitor (NNRTI) use, tenofovir use, ritonavir use, hepatocellular carcinoma, body mass index, and lowest CD4 count. Pre-LT 25(OH)D levels, post-LT routine vitamin D supplementation strategies, and post-LT vitamin D supplementation use were evaluated in post-LT analyses only. Potential predictors of 25(OH)D levels over the 2-year period after transplantation were examined via univariate and multivariate linear repeated measures models. In addition to the previously mentioned variables, the CD4 count, the MELD score, the glomerular filtration rate (estimated with the Chronic Kidney Disease Epidemiology Collaboration equation), HCV recurrence, the time after LT, and the aforementioned antiretroviral and vitamin D supplementation use variables were all evaluated as time-dependent covariates in the repeated measures models. In all analyses, variables with P < 0.15 from the univariate model were included in an initial multivariate model. Subsequently, variables with P ≥ 0.10 were excluded, the model was refit, and all interactions were examined. The 25(OH)D level at the initiation of HCV therapy [both as a binary factor (vitamin D deficiency or not) and as a continuous factor] was evaluated as a predictor of the end-of-treatment (EOT) response to therapy in a univariate logistic regression model. Despite the small sample size, the impact of the 25(OH)D level on the EOT rate was evaluated in a multivariate model adjusted for the HCV genotype, which was the only factor previously shown to be marginally associated with the EOT rate in this population. The 25(OH)D level was also evaluated as a time-dependent covariate in univariate proportional hazards regression models for post-LT death, the development of treated acute rejection, and histological HCV recurrence (in the HCV-infected subgroup only). The 25(OH)D level was examined as a binary factor (vitamin D deficiency or not and severe vitamin D deficiency or not) and as a continuous variable. A P value < 0.05 was considered statistically significant; all statistical analyses were performed with SAS 9.2 (SAS Institute, Cary, NC).
Prescription of Vitamin D Supplements
None of the centers routinely recommended vitamin D supplements before LT, and only a minority (4/17) recommended vitamin D supplements to all patients after LT (Supporting Table 2). At the 4 centers employing a strategy of routine supplementation after LT, the most commonly reported dose was 800 IU/day (3 centers). One center prescribed a vitamin D dose of 600 IU/day.
Vitamin D Status Before LT
One hundred thirty-nine candidates had baseline values: 48 (35%) had a severe deficiency, 98 (71%) had a deficiency, and 125 (90%) had levels < 32 ng/mL. None of the patients had 25(OH)D levels > 150 ng/mL, which is the level associated with vitamin D toxicity. The median 25(OH)D level was 12.6 ng/mL (IQR = 7.9-22.1 ng/mL; see Fig. 1). In a multivariate logistic regression model, cirrhosis (as the primary hepatic diagnosis) was the only factor significantly associated with vitamin D deficiency [odds ratio (OR) = 3.5, 95% confidence interval (CI) = 1.4-8.8, P = 0.01]; male sex was associated with a lower risk of deficiency, but the association was not statistically significant (OR = 0.3, 95% CI = 0.1-1.04, P = 0.06; Table 1).
Table 1. Factors Associated With Vitamin D Deficiency Before LT
Both pre- and post-LT samples were available for 83 patients. By 12 weeks after LT, the first time point analyzed, 25(OH)D levels had risen modestly (but significantly) to a median of 17.5 ng/mL (IQR: 12.3, 22.1 ng/mL; Fig. 1 and Table 2). The median increase was 4.5 ng/mL (IQR: −3.6, 11.8 ng/mL). Post-LT levels remained higher than pre-LT levels at all analyzed time points; however, 40% were vitamin D deficient 1 year after LT, and 44% were deficient 2 years after LT.
The 53 subjects with both baseline and 1-year 25(OH)D levels were included in univariate/multivariate logistic regression analyses to identify baseline factors that were associated with vitamin D deficiency and severe vitamin D deficiency 1 year after LT (see Supporting Tables 3 and 4, respectively). In the univariate analysis, black race and pre-LT 25(OH)D levels were both associated with vitamin D deficiency; however, in the multivariate analysis, only pre-LT 25(OH)D levels were significant (OR = 0.88 per ng/mL, 95% CI = 0.81-0.96, P = 0.004). In the univariate and multivariate analyses of baseline factors associated with severe vitamin D deficiency, the pre-LT 25(OH)D level was the only significant factor (OR = 0.87 per ng/mL, 95% CI = 0.78-0.97, P = 0.01).
Table 3. Univariate and Multivariate Linear Repeated Measures Analysis of Factors Associated With 25(OH)D Levels After LT
Estimate (95% CI)
Estimated with the Chronic Kidney Disease Epidemiology Collaboration equation.
Patient-reported data from concomitant medication logs.
−4.2 (−10.9 to 2.5)
−9.8 (−15.9 to −3.7)
5.7 (−4.7 to 16.1)
Cirrhosis as indication for LT
1.8 (−4.1 to 7.7)
2.1 (−3.4 to 7.6)
−1.1 (−6.7 to 4.6)
0.5 (−6.5 to 7.4)
Age at LT (per year)
−0.1 (−0.4 to 0.2)
MELD score at LT
0.02 (−0.2 to 0.3)
Pre-LT body mass index (per kg/m2)
−0.1 (−0.5 to 0.3)
Pre-LT lowest CD4 count (per 50 cells/μL)
0.3 (−0.6 to 1.2)
Pre-LT 25(OH)D level (per ng/mL)
Routine vitamin D supplementation strategy after LT
Table 4. Logistic Regression Analysis of the EOT Response to HCV Therapy
OR (95% CI)
25 (OH)D level at treatment initiation (per ng/mL)
Vitamin D deficiency at treatment initiation
HCV genotype 1
OR (95% CI)
25(OH)D level at treatment initiation (per ng/mL)
HCV genotype 1
In a multivariate linear repeated measures model of factors associated with post-LT 25(OH)D levels, a 10 ng/mL increase in the pre-LT 25(OH)D level was associated with an average increase of 6 ng/mL in the post-LT 25(OH)D level (P < 0.001); serum specimen collection in the winter and spring/fall was associated with 5.5 (P < 0.001) and 4.6 ng/mL decreases (P < 0.001), respectively, in comparison with collection in the summer; a strategy of routine vitamin D supplementation after LT was associated with a 9.5 ng/mL increase (P < 0.001); and the time elapsing after transplantation (every 13 weeks) was associated with a 0.7 ng/mL increase (P = 0.01). Black race was associated with a 6.1 ng/mL decrease in post-LT 25(OH)D levels (P = 0.02; Table 3).
Relationship Between 25(OH)D Levels and Outcomes for HCV-Positive Patients
The relationship between 25(OH)D levels and HCV recurrence after LT was examined in the subset of 67 subjects with known 25(OH)D values after LT or within the 3 months before LT (from the group of 89 HCV-infected LT recipients in the main study). Thirty-seven of these 67 subjects experienced HCV recurrence after LT. In a univariate proportional hazards regression analysis of HCV recurrence, vitamin D deficiency [hazard ratio (HR) = 0.56, 95% CI = 0.28-1.12, P = 0.10], severe vitamin D deficiency (HR = 1.29, 95% CI = 0.63-2.66, P = 0.48), and 25(OH)D levels (HR = 1.02 per ng/mL, 95% CI = 1.00-1.04, P = 0.09) after LT were not significantly associated with the incidence of HCV recurrence.
Response to Antiviral Treatment for HCV
The relationship between 25(OH)D levels and treatment outcomes was examined in 33 patients who were treated with interferon and ribavirin after LT and who had 25(OH)D levels measured within 6 months of the initiation of HCV therapy. Information about 25(OH)D levels was not available for another 6 patients who received HCV treatment.
Twenty-eight of the 33 patients were infected with HCV genotype 1, and 5 were infected with other genotypes. Seven of the 33 patients (21%) had an EOT response measured at week 48 of treatment. The median 25(OH)D level at treatment initiation was 30.3 ng/mL (IQR = 20.8-48.2 ng/mL) for those who achieved an EOT response and 17.8 ng/mL (IQR = 8.1-25.7 ng/mL) for those who did not (Fig. 2). A higher baseline 25(OH)D level was the only variable significantly associated with a higher EOT response rate in a multivariate logistic regression analysis (OR = 1.13 per ng/mL, 95% CI = 1.004-1.27, P = 0.04); HCV genotype 1 was marginally associated with a lower EOT response rate (OR = 0.08, 95% CI = 0.01-1.08, P = 0.06; Table 4).
Four of the 33 patients achieved an SVR. The median 25(OH)D level at the baseline was 39.3 ng/mL (IQR = 24.8-54.4 ng/mL) for those patients and 20.2 ng/mL (IQR = 9.8-25.7 ng/mL) for patients who did not have an SVR (Wilcoxon rank-sum test; P = 0.04). For the 2 patients with an SVR and HCV genotype 1, the baseline 25(OH)D levels were 30.3 and 48.2 ng/mL; for the 2 patients with an SVR and HCV genotype 2, the levels were 19.2 and 60.6 ng/mL.
Relationship Between 25(OH)D Levels, Acute Rejection, and Survival
There were 34 subjects with a treated acute rejection episode and 35 deaths in the subgroup of 95 LT recipients for whom 25(OH)D levels were known after LT or within the 3 months before LT. In univariate proportional hazards regression models of mortality, neither the 25(OH)D level nor the vitamin D status was significantly associated with death. In univariate models of treated acute rejection, neither the 25(OH) level nor the vitamin D status was significantly associated with the incidence of rejection episodes.
This study of patients enrolled in the Solid Organ Transplantation in HIV: Multi-Site Study is the first to examine 25(OH)D levels in HIV-positive patients before and after LT. The samples analyzed in this study were collected between November 2003 and October 2010 at 17 academic medical centers in the United States. We found that most of the patients had vitamin D deficiency before LT, and almost half had vitamin D deficiency after LT. Vitamin D deficiency was defined with a 25(OH)D cutoff of 20 ng/mL in agreement with the Institute of Medicine's determination that the health risks of vitamin D deficiency increase when 25(OH)D levels fall below this threshold. The high prevalence of vitamin D deficiency is striking because HIV practice guidelines recommend a 25(OH)D level > 32 ng/mL for HIV-positive patients, and higher 25(OH)D levels have been found to be predictive of posttransplant bone gain. Only 10% of the patients in our study had pre-LT 25(OH)D levels > 32 ng/mL. Among the variables that we examined, cirrhosis was the only factor significantly associated with an increased risk of vitamin D deficiency. This association between vitamin D deficiency and cirrhosis agrees with previous findings; however, when Venu et al. used a cutoff of 32 ng/mL to define vitamin D deficiency, they did not find an association with cirrhosis. We also observed a nonsignificant trend toward more vitamin D deficiency among women, which is consistent with the lower 25(OH)D levels found in women in other studies. Our findings highlight the need to pay particular attention to the vitamin D requirements of patients with cirrhosis and women when one is evaluating HIV-positive LT candidates and patients.
The variables that were not significantly associated with vitamin D deficiency before LT in this study included race, HCV as the etiology of liver disease, and NNRTI use. All the patients in the study were HIV-positive, and this precluded an analysis of HIV as a factor predisposing patients to vitamin D deficiency; however, other studies have not found an association between HIV infection and low 25(OH)D levels.[29-31] A number of previous cross-sectional studies have reported an association between NNRTIs and vitamin D deficiency,[30, 32-35] although this was not found in all studies. Some studies have specifically reported an association with efavirenz but not other NNRTIs, and this suggests that this may be a drug-specific effect rather than a class effect[32, 37, 38]; however the limited number of patients in this study did not allow for stratification based on specific NNRTIs. Our study did not find a significant association between ritonavir use and vitamin D deficiency. Ritonavir has previously been associated with higher levels of 25(OH)D in patients[30, 33] and impairments of vitamin D metabolism in vitro. HIV-positive patients may have an increased susceptibility to the ill effects of vitamin D deficiency if they are taking certain commonly used antiretroviral drugs; parathyroid hormone elevations occur in tenofovir users who have 25(OH)D levels < 30 ng/mL, and they likely increase the risk of bone loss and bone fracture even when 25(OH)D levels remain ≥ 20 ng/mL (which is the usual cutoff for vitamin D deficiency). Vitamin D supplements may still be especially important for HIV-positive patients taking tenofovir, as noted in the package insert.
None of the centers routinely recommended vitamin D supplements to all patients before LT, and only 4 of the 17 centers (24%) made this recommendation after LT. More extensive use of vitamin D supplements would almost certainly raise 25(OH)D levels in this population because the implementation of a routine supplementation strategy after LT increased 25(OH)D levels by an average of 9.5 ng/mL. A recent study found that among patients awaiting LT, the median increase in 25(OH)D was 6.0 ng/mL/1000 IU of vitamin D3/day.
An interesting finding in our study was the positive association between 25(OH)D levels and EOT responses among 33 HCV-positive patients who received pegylated interferon/ribavirin for recurrent HCV. The 4 patients who achieved an SVR had significantly higher 25(OH)D levels at the initiation of treatment in comparison with the patients who did not. These results are consistent with published data showing that vitamin D supplements increase SVR rates after LT. A prospective trial is needed to draw definitive conclusions about the impact of vitamin D supplements on HCV treatment responses in the LT population.
The strengths of our study include the analysis of data for HIV-positive patients undergoing LT at 17 academic medical centers throughout the United States and the ability to follow patients up to 2 years after LT with information about both clinical outcomes and 25(OH)D levels. The limitations include the lack of complete data about 25(OH)D levels and the lack of information about other indicators of vitamin D and bone status, such as the level of parathyroid hormone and bone density.
In summary, our study indicates that vitamin D deficiency is highly prevalent among HIV-positive patients who are candidates for LT and who undergo LT. Cirrhosis was strongly associated with vitamin D deficiency before LT. Many centers advised the use of vitamin D supplements after LT as indicated but usually not on a routine basis. A routine supplementation strategy was associated with a large increase (9.5 ng/mL) in 25(OH)D levels, and this indicates that supplements can be effective in this population. Clinical trials of vitamin D (and calcium) supplements are needed to determine the optimal doses of vitamin D supplements and their impact on clinical outcomes.
The authors thank the Solid Organ Transplantation in HIV: Multi-Site Study group, the study subjects, and the many individuals involved in the conduct and/or care of the patients enrolled in this study at each of the participating institutions.